In the past, the applicant's research has focused on aging, epidermal lipids, and the epidermal permeability barrier. This application reflects a significant change in direction for the PI, whose long-term objective is to study the effects of aging on epidermal stem cells. The short-term objective is to optimize a novel in vivo competitive repopulation assay for keratinocyte stem cells, in order to study the aging of stem cells. Studies of epidermal stem cells have been hampered by lack of markers by which to identify them, which in turn has been limited by lack of adequate in vivo stem cell assays. Studies of epidermal stem cells performed to other tissues have shown that in vitro colony forming units do not represent the true long-term repopulating cell. Borrowing from well-established functional assays in hemopoietic cells, we have designed a novel in vivo competitive repopulating assay to study stem cell function in epidermal cells. Our assay utilizes green fluorescent protein (GFP) to distinguish epidermally derived test cells from non-green competitor cells. These keratinocytes produce an epidermis when seeded in a chamber on the back of a non-GFP mouse. A limiting dilution technique allows the absolute number of stem cells in a keratinocyte population to be determined using this assay. This assay has two key features. The first is the use of a long-term competitive assay that ensures not only the detection of the most primitive epidermal stem cells but also the survival of the transplanted keratinocytes, even when seeded with very low numbers of test cells but also the survival of the transplanted keratinocytes, even when seeded with very low numbers of test cells. The second is the use of a limiting dilution design to allow stem cell quantification. The aim of these studies is first to fully optimize our assay, to enable us to assess aged and young epidermal stem cells. We will then compare the absolute number of stem cells from young vs. aged epidermis. These studies will be done in murine keratinocytes as well as in human keratinocytes. This work will allow us to begin to understand the changes in epidermal stem cells with age, and may point to further studies to improve epidermal cell function and/or wound healing in the aged. These studies will also provide the first in vivo assay for epidermal stem cells, an important step for analyzing putative markers for this elusive epidermal cell, for cutaneous stem cell-targeted gene therapy, and for more basic studies of epidermal stem cell regulation and differentiation.